GSM7474211: CHi-C D0 rep2; Mus musculus; OTHER
30-40 million cells/sample were fixed with 2% formaldehyde (Agar Scientific #AGR1026) for 10 min at room temperature, prior to harvesting from the tissue culture flasks. Digestion of cross-linked chromatin with HindIII (NEB #R3104), labelling with biotin-14-dATP (Invitrogen #19524016) and DNA Polymerase I (Large Klenow Fragment; NEB #M0210) and ligation (with T4 DNA ligase; Invitrogen #15224025) were all performed on nuclei kept intact. Chromatin cross-linking was then reversed by incubation with proteinase K (Roche #03115879001) at 65°C overnight and the DNA was purified by phenol/chloroform extraction. Aliquots of the Hi-C libraries were used to verify efficiency of chromatin digestion and digestion using standard PCR and qPCR. Then, biotin from un-ligated DNA ends was removed using T4 DNA polymerase (NEB #M0203) and the DNA was sonicated  (Covaris E220) to an average size of around 400 base pairs. Sonicated DNA was end-repaired using DNA Polymerase I (Large Klenow Fragment; NEB #M0210), T4 DNA polymerase (NEB #M0203) and T4 DNA polynucleotide kinase (NEB #M0201), then dATP was added to the 3′ ends of the DNA using Klenow exo- (NEB #M0212). The DNA was then subjected to double-sided SPRI bead size selection (AMPure XP beads; Beckman Coulter #A63881) and biotin-marked ligation products were isolated using MyOne Streptavidin C1 Dynabeads (Invitrogen #65002). After adapter ligation (Illumina PE PCR 1.0 and PE PCR 2.0 primers), the bead-bound Hi-C DNA was amplified with seven PCR amplification cycles. Promoter capture Hi-C was performed using a custom-made RNA capture bait system (Agilent Technologies) consisting of 39,021 individual biotinylated RNAs targeting the ends of 22,225 promoter-containing mouse HindIII restriction fragments, as described (PMID: 25752748). After a post-capture PCR (four amplification cycles using Illumina's PE PCR 1.0 and PE PCR 2.0 primers), the PCHi-C libraries were purified with AMPure XP beads (Beckman Coulter #A63881) and paired-end sequenced (HiSeq 1000, Illumina) at the Babraham Institute Sequencing Facility. Illumina TruSeq paired-end